US3127326A - Process for preparing g-amino- - Google Patents
Process for preparing g-amino- Download PDFInfo
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- US3127326A US3127326A US3127326DA US3127326A US 3127326 A US3127326 A US 3127326A US 3127326D A US3127326D A US 3127326DA US 3127326 A US3127326 A US 3127326A
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- 238000004519 manufacturing process Methods 0.000 title claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 50
- 108090000790 Enzymes Proteins 0.000 claims description 50
- NGHVIOIJCVXTGV-ALEPSDHESA-N 6-APA Chemical compound [O-]C(=O)[C@H]1C(C)(C)S[C@@H]2[C@H]([NH3+])C(=O)N21 NGHVIOIJCVXTGV-ALEPSDHESA-N 0.000 claims description 24
- 239000004365 Protease Substances 0.000 claims description 20
- 229940088598 Enzyme Drugs 0.000 claims description 16
- 108091005771 Peptidases Proteins 0.000 claims description 14
- 102000035443 Peptidases Human genes 0.000 claims description 10
- 239000011541 reaction mixture Substances 0.000 claims description 10
- 241000588724 Escherichia coli Species 0.000 claims description 6
- 241000588767 Proteus vulgaris Species 0.000 claims description 6
- 229940007042 Proteus vulgaris Drugs 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 4
- 230000002797 proteolythic Effects 0.000 claims description 4
- MDQGTDMBVJCTBN-JCGDXUMPSA-N 6-formamidopenicillanic acid Chemical compound OC(=O)[C@H]1C(C)(C)S[C@@H]2[C@H](NC=O)C(=O)N21 MDQGTDMBVJCTBN-JCGDXUMPSA-N 0.000 claims 2
- 239000000243 solution Substances 0.000 description 52
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 44
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 34
- 229940110715 ENZYMES FOR TREATMENT OF WOUNDS AND ULCERS Drugs 0.000 description 32
- 229940020899 hematological Enzymes Drugs 0.000 description 32
- 150000002960 penicillins Chemical class 0.000 description 32
- YXFVVABEGXRONW-UHFFFAOYSA-N toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 32
- 229940049954 Penicillin Drugs 0.000 description 28
- 229960000626 benzylpenicillin Drugs 0.000 description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 24
- 229940043232 butyl acetate Drugs 0.000 description 22
- 238000000034 method Methods 0.000 description 18
- 229940056367 Penicillin V Drugs 0.000 description 12
- BPLBGHOLXOTWMN-MBNYWOFBSA-N Phenoxymethylpenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)COC1=CC=CC=C1 BPLBGHOLXOTWMN-MBNYWOFBSA-N 0.000 description 12
- 239000007864 aqueous solution Substances 0.000 description 12
- 235000018102 proteins Nutrition 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 229940055729 Papain Drugs 0.000 description 10
- 108090000526 Papain Proteins 0.000 description 10
- 239000002253 acid Substances 0.000 description 10
- 238000000605 extraction Methods 0.000 description 10
- 235000019834 papain Nutrition 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- 241000233866 Fungi Species 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 229940056360 Penicillin G Drugs 0.000 description 6
- 241000228150 Penicillium chrysogenum Species 0.000 description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 6
- 239000012062 aqueous buffer Substances 0.000 description 6
- 238000003379 elimination reaction Methods 0.000 description 6
- 238000004108 freeze drying Methods 0.000 description 6
- 159000000001 potassium salts Chemical class 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 108090000317 Chymotrypsin Proteins 0.000 description 4
- 102000033147 ERVK-25 Human genes 0.000 description 4
- 241000228143 Penicillium Species 0.000 description 4
- 229940024999 Proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 4
- 229960004319 Trichloroacetic Acid Drugs 0.000 description 4
- YNJBWRMUSHSURL-UHFFFAOYSA-N Trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 229960002376 chymotrypsin Drugs 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000001953 recrystallisation Methods 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 159000000000 sodium salts Chemical class 0.000 description 4
- 229960001322 trypsin Drugs 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- MXKBHIPOOQSROI-UHFFFAOYSA-N 3-bromo-4-tert-butylaniline;hydrochloride Chemical compound Cl.CC(C)(C)C1=CC=C(N)C=C1Br MXKBHIPOOQSROI-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 102000005600 Cathepsins Human genes 0.000 description 2
- 108010084457 Cathepsins Proteins 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- 101700065453 LAP3 Proteins 0.000 description 2
- 210000000496 Pancreas Anatomy 0.000 description 2
- RBKMMJSQKNKNEV-RITPCOANSA-N Penicillanic acid Chemical compound OC(=O)[C@H]1C(C)(C)S[C@@H]2CC(=O)N21 RBKMMJSQKNKNEV-RITPCOANSA-N 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 230000001580 bacterial Effects 0.000 description 2
- 230000005591 charge neutralization Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 150000001805 chlorine compounds Chemical class 0.000 description 2
- 230000000875 corresponding Effects 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 239000012045 crude solution Substances 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000002255 enzymatic Effects 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- AMQJEAYHLZJPGS-UHFFFAOYSA-N n-pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 description 2
- 230000001264 neutralization Effects 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 210000000056 organs Anatomy 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 235000013311 vegetables Nutrition 0.000 description 2
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D499/00—Heterocyclic compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. penicillins, penems; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/832—Bacillus
- Y10S435/839—Bacillus subtilis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/848—Escherichia
- Y10S435/849—Escherichia coli
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/873—Proteus
Definitions
- the various penicillins can be prepared from this 6- arnino-penicillanic acid by reaction with the corresponding acid chlorides.
- 6-amino-penici1lanic acid has recently been obtained according to research Work carried out by F. R. Batchelor, F. P. Doyle, J. H. C. Nayler and G. N. Robinson (Nature, vol. 183, -No. 465 6, Jan. 24, 1959, from fermentation solutions of Penicillium chrysogenum W. 51.20.
- 6-amino-penicillanic acid can be obtained by causing proteolytic enzymes to act on penicillins and isolating the 6-amino-penicillanic acid from the reaction mixture, if necessary after separation of the penicillin that has not reacted.
- penicillins there enter into consideration for example penicillin G or penicillin 0 V. It is possible to start from pure penicillins or their salts or from penicillins in the form of the crude culture solutions or from the concentrations obtained in the process of manufacture in aqueous buifer solutions.
- proteolytic enzymes there are suitable, for in- 5 stance, trypsin, chymotrypsin, polypeptidases, papain, fungus proteases, the animal and vegetable cathepsins as well as pepsin.
- the various enzymes can be used in pure form as well as in the form of crude products, for instance organor fungus dry preparations or crude solutions.
- a pancreatic extract is suitable as organ extract.
- the enzyme action on the solutions of the penicillin salts is eifected at the temperatureand pH-conditions entering into consideration for the individual enzymes.
- the penicillin that. has not reacted is favorably extracted, While showing an acid reaction, from the solution by means of organic solvents and the 6-amino-penicillanic acid is isolated from the aqueous mother liquor according to known methods. It has proved advantageous to inactivate or to remove the enzymes prior to the further treatment.
- Example 1 0.5 gram of the potassium salt of penicillin V is dissolved in 25 cc. of water and mixed with a solution of milligrams of crude papain in 25 cc. of water, activated by cysteine.
- the pH-value of the batch is adjusted to 5.0 by means of dilute hydrochloric acid and the batch is incubated again for 12 hours at 40 C. with protection by toluene and with constant pH-value in the water bath.
- the pH-value is adjusted to 2 by means of dilute sulfuric acid and penicillin that has not reacted is removed by double extraction by means of butyl-acetate.
- the residual neutralized aqueous solution is subjected to lyophilisation and from the dry product thus obtained there is extracted and isolated the G-aminopenicillanic acid by means of methanol.
- Example 3 0.5 gram of the sodium salt of penicillin G is dissolved in 25 cc. of water and mixed with 200 milligrams of a dry preparation of fungus protease which had been dissolved in 25 cc. of water.
- the pH-value of the solution is adjusted to 5.3 and maintained by occasional addition of dilute sodium hydroxide solution during the splitting by the enzymes which is carried out within 12 hours with protection by toluene in the water bath at ⁇ +37 C.
- the reaction mixture is then adjusted to a pH-value of 2 by means of dilute sulfuric acid and penicillin that has not reacted is removed by shaking it out twice with butylacetate.
- the elimination of protein is carried out in 'known manner by means of trichloroacetic acid. In the remaining aqueous phase the 6-amino-penicillanic acid formed is identified in the bioautogram after neutralisation.
- the dry preparation of fungus protease can be obtained, for instance, by:
- Example 4 To 1 liter of a culture filtrate solution of Penicillz'um chrysogenum with 7,000 units of penicillin V/milliliter there is added a solution of 1 gram of crude papain in 100 cc. of water. The solution is adjusted to pH 6.5 and split by the enzymes within several hours with protection of toluene at 40 C. while maintaining this pH-value. When splitting by the enzymes is finished the pH-value is adjusted to 2.0 by means of acid and the penicillin that has not reacted is removed by extraction with 0.5 liter of butyl-acetate. In order to remove the enzyme and ballast protein substances still contained in the residual aqueous solution, the solution is treated in the usual man her by means of trichloracetic acid. The solution free from protein is neutralised, the solution containing the 6-amino-penicillanic acid is lyophilized and the dry preparation is worked up to pure 6-amino-penicillanic acid by extraction with alcohol.
- Example 5 4 liters of a culture filtrate solution of Penicillium chrysogenum of 7,000 units of penicillin V/milliliter are shaken out with 2 liters of butyl-acetate. After separation of the butyl-acetate phase containing penicillin the latter is extracted with 200 cc. of see-sodium phosphate in an aqueous solution of 2% strength.
- the aqueous buffer solution containing penicillin after separation from the butyl-acetate is adjusted to a pH- value of 6.5 and treated with protection by toluene with 4 grams of a mixture of equal parts of chymotrypsin and trypsin.
- the spitting by the enzymes is carried out within 12 hours at 40 C. while maintaining the pH-value.
- Example 6 0.5 gram of the sodium salt of penicillin G is dissolved in 50 milliliters of water and a strongly enzymatic cell homogenate from 0.5 gram of E. coli dry cells is added. The mixture is incubated for 12 hours at a pH-value of 7.5 to 8.0 at 40 C. with protection by toluene. The penicillin that has not reacted is then removed at a pH- value of 2.0 by twice shaking it out with 25 milliliters of butyl-acetate each time. The residual solution is adjusted to a pH-value of 6.0, freed, in known manner, from protein and concentrated under reduced pressure to about 25 milliliters. A pI-I-value of 4.3 is adjusted by means of concentrated hydrochloric acid.
- Example 7 0.5 gram of the potassium salt of penicillin V is added to milliliters of a culture filtrate from a 14 to 28 day old culture of Proteus vulgaris and the enzymes contained therein are allowed to act with protection by toluene for 12 hours at a pH-value of 7.5 to 8.0 and at 40 C.
- the penicillin that has not been split by the added bacterial enzymes is then removed by shaking it out with butyl-acetate and after preliminary elimination of protein the penicillanic acid that has formed is obtained from the neutral solution in the manner described in Example 6 by strong concentration under reduced pressure and adjustment to a pH-value of 4.3.
- the recrystallisation is likewise carried out according to Example 6.
- Example 8 4 liters of a culture filtrate of Penicillium clzrysogenum with an activity of 7,000 units/milliliter are extracted with 2.0 liters of butyl-acetate at a pH-value of 2.0. The extract is shaken back with 200 milliliters of phosphate buffer (2% strength, Na HPO After elimination of the residual butyl-acetate 100 milliliters of an about 4 week old, autolysed culture of Bac. subtilis is added. After an action of 18 hours of the enzymes contained in the autolysate at a pH-value of 7 .5 to 8.0 at 40 C. with protection by toluene the penicillin that did not react is removed by extraction and the 6-amino-penicillanic acid remained in the residual solution is isolated as described in Example 6.
- a process for preparing G-amino-penicillanic acid which comprises causing a proteolytic enzyme selected from the group consisting of papain and proteases obtained from the microorganisms E. coli, Proteus vulgaris, and Bac. subtilis, to act on penicillins, and then isolating 6-amino-penicillanic acid from the reaction mixture.
- a proteolytic enzyme selected from the group consisting of papain and proteases obtained from the microorganisms E. coli, Proteus vulgaris, and Bac. subtilis
- 6-amino-penicillanic acid which comprises treating an aqueous solution of a biosynthetically prepared penicillin with an aqueous solution of papain at a pH of 5 and at a temperature of about 37 C.
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
United States Patent ()fiice 3,127,326 Patented Mar. 31, 1964 The various known penicillins possess the following chemical basic structure:
and differentiate from each other only by the grouping designated by R; for instance in the case of- 6-amino-penicillanic acid of the following constitution 35 is believed to be the common basic substance of all penicillins:
Penicillin V:
The various penicillins can be prepared from this 6- arnino-penicillanic acid by reaction with the corresponding acid chlorides.
6-amino-penici1lanic acid has recently been obtained according to research Work carried out by F. R. Batchelor, F. P. Doyle, J. H. C. Nayler and G. N. Robinson (Nature, vol. 183, -No. 465 6, Jan. 24, 1959, from fermentation solutions of Penicillium chrysogenum W. 51.20.
Now we have found that 6-amino-penicillanic acid can be obtained by causing proteolytic enzymes to act on penicillins and isolating the 6-amino-penicillanic acid from the reaction mixture, if necessary after separation of the penicillin that has not reacted. As penicillins there enter into consideration for example penicillin G or penicillin 0 V. It is possible to start from pure penicillins or their salts or from penicillins in the form of the crude culture solutions or from the concentrations obtained in the process of manufacture in aqueous buifer solutions.
As proteolytic enzymes there are suitable, for in- 5 stance, trypsin, chymotrypsin, polypeptidases, papain, fungus proteases, the animal and vegetable cathepsins as well as pepsin. The various enzymes can be used in pure form as well as in the form of crude products, for instance organor fungus dry preparations or crude solutions. For instance, a pancreatic extract is suitable as organ extract.
The enzyme action on the solutions of the penicillin salts is eifected at the temperatureand pH-conditions entering into consideration for the individual enzymes. Upon action of the enzyme the penicillin that. has not reacted is favorably extracted, While showing an acid reaction, from the solution by means of organic solvents and the 6-amino-penicillanic acid is isolated from the aqueous mother liquor according to known methods. It has proved advantageous to inactivate or to remove the enzymes prior to the further treatment. For this stage of the process there enter into consideration, for example, the precipitation with reagents for the precipitation of protein or the treatment with organic solvents according to Sevag.
As already mentioned, there can be used as starting product apart from pure crystallized penicillin salts likewise favorably the culture solutions at first obtained in the production of penicillin or, in aqueous buffer solutions, the solutions of penicillins obtained in the recovery process.
The following examples serve to illustrate the invention but they are not intended to limit it thereto.
Example 1 0.5 gram of the potassium salt of penicillin V is dissolved in 25 cc. of water and mixed with a solution of milligrams of crude papain in 25 cc. of water, activated by cysteine. The pH-value of the batch is adjusted to 5.0 by means of dilute hydrochloric acid and the batch is incubated again for 12 hours at 40 C. with protection by toluene and with constant pH-value in the water bath. When splitting is finished, the pH-value is adjusted to 2 by means of dilute sulfuric acid and penicillin that has not reacted is removed by double extraction by means of butyl-acetate. The residual neutralized aqueous solution is subjected to lyophilisation and from the dry product thus obtained there is extracted and isolated the G-aminopenicillanic acid by means of methanol.
Example 2 0.5 gram of the potassium salt of penicillin V is dissolved in 48 cc. of water and an active enzyme juice obtained by slow thawing of a frozen cattle pancreas is added. After a pH-value of 6.8 has been adjusted, the reaction mixture is introduced into the water bath of 40 C. and split by the enzyme with protection by toluene for 12 hours while a pH of 6. 8 is maintained. The mixture is then adjusted to pH=2 by means of dilute sulfuric acid and the unreacted penicillin is removed by shaking it out twice with 30 cc. of butyl-acetate each. In order to remove the enzyme protein, the aqueous solution is treated according to the Sevag process by shaking it out with a mixture of chloroform and amyl alcohol.
The further treatment is carried out as described in Example 1.
Example 3 0.5 gram of the sodium salt of penicillin G is dissolved in 25 cc. of water and mixed with 200 milligrams of a dry preparation of fungus protease which had been dissolved in 25 cc. of water. The pH-value of the solution is adjusted to 5.3 and maintained by occasional addition of dilute sodium hydroxide solution during the splitting by the enzymes which is carried out within 12 hours with protection by toluene in the water bath at {+37 C. The reaction mixture is then adjusted to a pH-value of 2 by means of dilute sulfuric acid and penicillin that has not reacted is removed by shaking it out twice with butylacetate. The elimination of protein is carried out in 'known manner by means of trichloroacetic acid. In the remaining aqueous phase the 6-amino-penicillanic acid formed is identified in the bioautogram after neutralisation. The dry preparation of fungus protease can be obtained, for instance, by:
(1) Lyophilisation of the culture solution of, for example, Penicillium chrysogerzum,
(2) By disintegration of the mycelium of, for example, Penicillium chrysogenum, extraction and lyophilisation of the solution. There can likewise be used the enzymes further purified by fractionation of alcohol.
Example 4 To 1 liter of a culture filtrate solution of Penicillz'um chrysogenum with 7,000 units of penicillin V/milliliter there is added a solution of 1 gram of crude papain in 100 cc. of water. The solution is adjusted to pH 6.5 and split by the enzymes within several hours with protection of toluene at 40 C. while maintaining this pH-value. When splitting by the enzymes is finished the pH-value is adjusted to 2.0 by means of acid and the penicillin that has not reacted is removed by extraction with 0.5 liter of butyl-acetate. In order to remove the enzyme and ballast protein substances still contained in the residual aqueous solution, the solution is treated in the usual man her by means of trichloracetic acid. The solution free from protein is neutralised, the solution containing the 6-amino-penicillanic acid is lyophilized and the dry preparation is worked up to pure 6-amino-penicillanic acid by extraction with alcohol.
Example 5 4 liters of a culture filtrate solution of Penicillium chrysogenum of 7,000 units of penicillin V/milliliter are shaken out with 2 liters of butyl-acetate. After separation of the butyl-acetate phase containing penicillin the latter is extracted with 200 cc. of see-sodium phosphate in an aqueous solution of 2% strength.
The aqueous buffer solution containing penicillin after separation from the butyl-acetate is adjusted to a pH- value of 6.5 and treated with protection by toluene with 4 grams of a mixture of equal parts of chymotrypsin and trypsin. The spitting by the enzymes is carried out within 12 hours at 40 C. while maintaining the pH-value.
The further treatment is carried out according to Example 4.
Example 6 0.5 gram of the sodium salt of penicillin G is dissolved in 50 milliliters of water and a strongly enzymatic cell homogenate from 0.5 gram of E. coli dry cells is added. The mixture is incubated for 12 hours at a pH-value of 7.5 to 8.0 at 40 C. with protection by toluene. The penicillin that has not reacted is then removed at a pH- value of 2.0 by twice shaking it out with 25 milliliters of butyl-acetate each time. The residual solution is adjusted to a pH-value of 6.0, freed, in known manner, from protein and concentrated under reduced pressure to about 25 milliliters. A pI-I-value of 4.3 is adjusted by means of concentrated hydrochloric acid. After standing for some time in the cold 6-amino-penicillanic acid separates off and after filtering with suction is washed with water and acetone; it is then dried under reduced pressure. The recrystallization is carried out by dissolution in water at a pH-value of 7.0 and reacidification by means of hydrochlorid acid. Melting point 208 C. to 209 C.
4 Example 7 0.5 gram of the potassium salt of penicillin V is added to milliliters of a culture filtrate from a 14 to 28 day old culture of Proteus vulgaris and the enzymes contained therein are allowed to act with protection by toluene for 12 hours at a pH-value of 7.5 to 8.0 and at 40 C.
The penicillin that has not been split by the added bacterial enzymes is then removed by shaking it out with butyl-acetate and after preliminary elimination of protein the penicillanic acid that has formed is obtained from the neutral solution in the manner described in Example 6 by strong concentration under reduced pressure and adjustment to a pH-value of 4.3. The recrystallisation is likewise carried out according to Example 6.
Example 8 4 liters of a culture filtrate of Penicillium clzrysogenum with an activity of 7,000 units/milliliter are extracted with 2.0 liters of butyl-acetate at a pH-value of 2.0. The extract is shaken back with 200 milliliters of phosphate buffer (2% strength, Na HPO After elimination of the residual butyl-acetate 100 milliliters of an about 4 week old, autolysed culture of Bac. subtilis is added. After an action of 18 hours of the enzymes contained in the autolysate at a pH-value of 7 .5 to 8.0 at 40 C. with protection by toluene the penicillin that did not react is removed by extraction and the 6-amino-penicillanic acid remained in the residual solution is isolated as described in Example 6.
We claim:
1. A process for preparing G-amino-penicillanic acid which comprises causing a proteolytic enzyme selected from the group consisting of papain and proteases obtained from the microorganisms E. coli, Proteus vulgaris, and Bac. subtilis, to act on penicillins, and then isolating 6-amino-penicillanic acid from the reaction mixture.
2. A process as in claim 1 wherein a member of the group consisting of pure penicillins and the salts of penicillins is used as starting substance.
3. A process as in claim 1 wherein said penicillins are reacted with said enzymes in the culture solutions for said penicillins.
4. A process as in claim 1 wherein said penicillins are reacted with said enzymes in aqueous buffer solutions.
5. A process as in claim 1 wherein said enzymes are reacted in unrefined dry form.
6. A process as in claim 1 wherein said enzymes are reacted in the form of unrefined enzyme extracts.
7. The process for the preparation of 6-amino-penicillanic acid which comprises treating an aqueous solution of a biosynthetically prepared penicillin with an aqueous solution of papain at a pH of 5 and at a temperature of about 37 C.
References Cited in the file of this patent Nature; vol. 183, pages 257-258, January 24, 1959. (Copy in P.O.S.L.)
J. Agr. Chem. Soc. Japan, 23, page 411 (1950). (Copy in P.O.S.L.)
Enzymes, Waksman & Davidson, pages -118, 1926, published by Williams & Wilkins.
Claims (1)
1. A PROCESS FOR PREPARING 6-AMINO-PENICILLANIC ACID WHICH COMPRISES CAUSING A PROTEOLYTIC ENZYME SELECTED FROM THE GROUP CONSISTING OF PAPAIN AND PROTEASES OBTAINED FROM THE MICROORGANISMS E. COLI, PROTEUS VULGARIS, AND BAC, SUBTILIS, TO ACT ON PENICILLINS, AND THEN ISOLATING 6-AMINO-PENICILLANIC ACID FROM THE REACTION MIXTURE.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| US3127326A true US3127326A (en) | 1964-03-31 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US3127326D Expired - Lifetime US3127326A (en) | Process for preparing g-amino- |
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| Country | Link |
|---|---|
| US (1) | US3127326A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3260653A (en) * | 1959-04-18 | 1966-07-12 | Bayer Ag | Process for the production of 6-aminopenicillanic acid |
| US3297546A (en) * | 1963-12-09 | 1967-01-10 | Bristol Myers Co | Preparation of 6-aminopenicillanic acid |
-
0
- US US3127326D patent/US3127326A/en not_active Expired - Lifetime
Non-Patent Citations (1)
| Title |
|---|
| None * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3260653A (en) * | 1959-04-18 | 1966-07-12 | Bayer Ag | Process for the production of 6-aminopenicillanic acid |
| US3297546A (en) * | 1963-12-09 | 1967-01-10 | Bristol Myers Co | Preparation of 6-aminopenicillanic acid |
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